RESUMEN
Site-directed mutagenesis has been used to change three amino acid residues involved in the binding of inhibitors (Asn67Ile; Gln92Val and Leu204Ser) within the active site of human carbonic anhydrase (CA, EC 4.2.1.1) II (hCA II). Residues 67, 92 and 204 were changed from hydrophobic to hydrophilic ones, and vice versa. The Asn67Ile and Leu204Ser mutants showed similar k(cat)/K(M) values compared to the wild type (wt) enzyme, whereas the Gln92Val mutant was around 30% less active as a catalyst for CO(2) hydration to bicarbonate compared to the wt protein. Affinity for sulfonamides/sulfamates was decreased in all three mutants compared to wt hCA II. The effect was stronger for the Asn67Ile mutant (the closest residue to the zinc ion), followed by the Gln92Val mutant (residue situated in the middle of the active site) and weakest for the Leu204Ser mutant, an amino acid situated far away from the catalytic metal ion, at the entrance of the cavity. This study shows that small perturbations within the active site architecture have influences on the catalytic efficiency but dramatically change affinity for inhibitors among the CA enzymes, especially when the mutated amino acid residues are nearby the catalytic metal ion.
Asunto(s)
Anhidrasa Carbónica II , Inhibidores de Anhidrasa Carbónica/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Dominio Catalítico , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO(2) hydration reaction were measured. With a k(cat)/K(m) of 1.1 × 10(8) M(-1) s(-1), and a k(cat) of 1.3 × 10(6) s(-1), clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K(I)s in the range of 1.9-3460 nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Animales , Aves , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/metabolismo , Activación Enzimática/efectos de los fármacos , Eritrocitos/enzimología , Estructura Molecular , Isoformas de ProteínasRESUMEN
Hypoxia is a common characteristic of locally advanced solid tumors that has been associated with diminished therapeutic response and malignant progression. Human carbonic anhydrase (hCA) hCA IX and XII isozymes are tumor associated isoforms which contribute to acidification of the tumor environment by catalyzing the hydration of carbon dioxide to bicarbonate and protons.In the present study our goal was to investigate the inhibition effects of 15 different antibiotics belonging to the following classes: Lactams, cephalosporins, macrolides etc., on the tumor associated carbonic anhydrase isozymes hCA-IX, hCA-XII and cytosolic carbonic anhydrase hCA-I and hCA-II.
Asunto(s)
Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Antígenos de Neoplasias/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Anhidrasa Carbónica IX , Cefalosporinas/química , Cefalosporinas/farmacología , Humanos , Isoenzimas/metabolismo , Lactamas/química , Lactamas/farmacología , Macrólidos/química , Macrólidos/farmacología , Neoplasias/enzimologíaRESUMEN
A new series of 6, 7-dihydroxy-3-(methylphenyl) chromenones, including three new derivatives, i.e. 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC); 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC); 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) and one previously described, namely 6,7-dihydroxy-3-phenyl-2H-chromen-2-one (DPC), were synthesized. These compounds were investigated as inhibitors of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which had been purified from human erythrocytes on an affinity gel comprised of L-tyrosine-sulfonamide-Sepharose 4B.
Asunto(s)
Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica I/antagonistas & inhibidores , Cumarinas/farmacología , Inhibidores Enzimáticos/farmacología , Citosol/enzimología , Humanos , Hidroxilación , CinéticaRESUMEN
The enzyme carbonic anhydrase (E.C. 4.2.1.1) has a stimulatory effect on glaucoma, an eye disease that has a risk to dogs, which are models for the human eye disease, that is similar to that in humans. In this study, some sulfonamide derivatives, 2-(3-cyclohexene-1-carbamido)-1,3,4-thiadiazole-5-sulfonamide (CCTS), 4-(3-cyclohexene-1-carbamido) methyl-benzenesulfonamide (CCBS), 2-(9-octadecenoylamido)-1,3,4-thiadiazole-5-sulfonamide (ODTS), 2-(4,7,10-trioxa-tetradecanoylamido)-1,3,4-thiadiazole-5-sulfonamide (TDTS), and 2-(8-methoxycoumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (MCTS), as well as some anionic compounds (perchlorate and chloride) and existing medicines (dorzolamide-HCl, gentamicine sulphate, tropicamide, and procaine-HCl) were assayed for their inhibition of dog carbonic anhydrase (dCA), which was purified from erythrocytes on an affinity gel of L-tyrosine-sulfonamide-Sepharose 4B. ODTS showed the highest potency amongst the synthetic compounds with IC50 value 1.18 x 10(-5) M. Amongst the medicines tested, only dorzolamide showed inhibition with IC50 value 5.05 x 10(-4) M. Procaine and tropicamide actually showed an activatory effect, whereas gentamicine sulfate had no significant effect. The inhibitory effects of anionic compounds such as perchlorate and chloride were also investigated; whereas perchlorate showed inhibition, chloride did not.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/aislamiento & purificación , Anhidrasas Carbónicas/metabolismo , Eritrocitos/enzimología , Animales , Inhibidores de Anhidrasa Carbónica/química , Cromatografía de Afinidad , Perros , Concentración 50 Inhibidora , Estructura MolecularRESUMEN
OBJECTIVES: The antioxidant enzyme catalase and the CO2/HCO3- exchange enzyme carbonic anhydrase are both present in significant amounts in the cytosol of erythrocytes. The aim of the present study was to investigate whether these erythrocyte enzyme activities are altered in patients who have carcinoma. DESIGN AND METHODS: Cytosolic erythrocyte enzyme activities were measured in 108 cancer patients presenting with carcinoma at one of variable sites, prior to clinical treatment. These were compared with an age- and sex-matched control group of 31 healthy volunteers. RESULTS: Mean (+/-SD) catalase activities did not differ significantly, i.e. 0.0035 (+/-0.0015) EU/ml in carcinoma patients vs. 0.0031 (+/-0.00075) EU/ml in controls. However, mean carbonic anhydrase activities of 204 (+/-91) EU/ml in the carcinoma patients were significantly higher than the 158 (+/-35) EU/ml in controls (P value of 0.0065). CONCLUSION: Cytosolic erythrocyte carbonic anhydrase levels may warrant further investigation as a potential peripheral marker in cancer.
Asunto(s)
Anhidrasas Carbónicas/sangre , Catalasa/sangre , Citosol/enzimología , Eritrocitos/enzimología , Neoplasias/enzimología , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Neoplasias/sangreRESUMEN
The purification of red blood cell carbonic anhydrase (CA, EC 4.2.1.1) from ostrich (scCA) blood is reported, as well as an inhibition study of this enzyme with a series of aromatic and heterocylic sulfonamides. The ostrich enzyme showed a high activity, comparable to that of the human isozyme II, with kcat, of 1.2 x 10(6) s(-1) and kcat/KM of 1.8 x 10(7) M(-1)s(-1), and an inhibition profile quite different from that of the human red blood cell cytosolic isozymes hCA I and II. scCA has generally a lower affinity for sulfonamide inhibitors as compared to hCA I and II. The only sulfonamide which behaved as a very potent inhibitor of this enzyme was ethoxzolamide (KI = 3.9 nM) whereas acetazolamide and sulfanilamide behaved as weaker inhibitors (inhibition constants in the range 303-570 nM). Several other aromatic and heterocyclic sulfonamides, mostly derivatives of sulfanilamide, homosulfanilamide, 4-aminoethylbenzenesulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide, showed good affinities for the ostrich enzyme, with KI values in the range 25-72 nM.
Asunto(s)
Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica I/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Eritrocitos/enzimología , Struthioniformes/metabolismo , Sulfonamidas/farmacología , Animales , Inhibidores de Anhidrasa Carbónica/química , Femenino , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
The inhibition of the two transmembrane, tumor-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1) of human origin, hCA IX and XII, with a library of aromatic and heteroaromatic sulfonamides has been investigated. Most of them were sulfanilamide, homosulfanilamide, and 4-aminoethyl-benzenesulfonamide derivatives, to which tails that should induce diverse physico-chemical properties have been attached at the amino moiety, whereas several of these compounds were derived from metanilamide, benzene-1,3-disulfonamide or the 1,3,4-thiadiazole/thiadiazoline-2-sulfonamides. The tails were of the alkyl/aryl-carboxamido/sulfonamido-, ureido or thioureido type. Against hCA IX the investigated compounds showed inhibition constants in the range of 3-294 nM, whereas against hCA XII in the range of 1.9-348 nM, respectively. The best hCA IX inhibitors were ureas/thioureas incorporating 4-aminoethyl-benzenesulfonamide and metanilamide moieties. The best hCA XII inhibitors were 1,3,4-thiadiazole/thiadiazoline-2-sulfonamides incorporating 5-acylamido or 5-arylsulfonylamido moieties. These compounds also inhibited appreciably the cytosolic isozymes hCA I and II, but some selectivity for the transmembrane, tumor-associated isozymes was observed for some of them, which is an encouraging result for the design of novel therapies targeting hypoxic tumors, in which these carbonic anhydrases are highly overexpressed.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/química , Proteínas de Neoplasias/antagonistas & inhibidores , Sulfonamidas/farmacología , Antígenos de Neoplasias/efectos de los fármacos , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/efectos de los fármacos , Humanos , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
The first inhibition study of the transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA XIV with a library of aromatic and heteroaromatic sulfonamides synthesized earlier is reported. Most of the inhibitors were sulfanilamide, homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives, to which tails that would induce diverse physicochemical properties have been attached at the amino moiety. Several of these compounds were metanilamide, benzene-1,3-disulfonamide or the 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide derivatives. The tails incorporated in these molecules were of the alkyl/aryl-carboxamido/ sulfonamido-, ureido- or thioureido-types. The sulfanilamides acylated at the 4-amino group with short aliphatic/aromatic moieties incorporating 2-6 carbon atoms showed modest hCA XIV inhibitory activity (K(I)-s in the range of 1.25-4.2 microM) which were anyhow better than that of sulfanilamide (K(I) of 5.4 microM). Better activity showed the homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives bearing arylsulfonamido/ureido and thioureido moieties, with K(I)'s in the range of 203-935 nM. The best activity was observed for the heteroaromatic compounds incorporating 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide and 5-arylcarboxamido/sulfonamido moieties, with K(I)'s in the range of 10-85 nM. All these compounds were generally also much better inhibitors of the other two transmembrane CA isozyme, hCA IX and XII. Thus, highly potent hCA XIV inhibitors were detected, but isozyme-specific inhibitors were not discovered for the moment.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/efectos de los fármacos , Compuestos Heterocíclicos/química , Hidrocarburos Aromáticos/química , Sulfonamidas/química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/genética , Compuestos Heterocíclicos/clasificación , Compuestos Heterocíclicos/farmacología , Humanos , Hidrocarburos Aromáticos/clasificación , Hidrocarburos Aromáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Relación Estructura-Actividad , Sulfonamidas/clasificación , Sulfonamidas/farmacologíaRESUMEN
A new series of aromatic and heterocyclic sulfonamides, including six new derivatives, 2-(3-cyclohexene-1-carbamido)-1,3,4-thiadiazole-5-sulfonamide (CCTS), 4-(3-cyclohexene-1-carbamido) methyl-benzenesulfonamide (CCBS), 2-(9-octadecenoylamido)-1,3,4-thiadiazole-5-sulfonamide (ODTS), 2-(4,7,10-trioxa-tetradecanoylamido)-1,3,4-thiadiazole-5-sulfonamide (TDTS), 2-(coumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (COTS) and 2-(8-methoxycoumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (MCTS), has been investigated. These sulfonamides were assayed for inhibition of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which were purified by affinity chromatography.